Light and storage on the germination of spores of Dicksonia sellowiana (Presl.) Hook., Dicksoniaceae

Luz e armazenamento na germinação de esporos de Dicksonia sellowiana (Presl.) Hook., Dicksoniaceae

Light and storage on the germination of spores of Dicksonia sellowiana (Presl.) Hook., Dicksoniaceae

ELAINE CRISTINA DE PAULA FIILIPPINI¹, SONIA REGINA DUZ¹ and ÁUREA MARIA RANDI¹

(recebido em 20/04/98; aceito em 18/09/98)

ABSTRACT - (Light and storage on the germination of spores of Dicksonia sellowiana (Presl.) Hook., Dicksoniaceae). Spores of Dicksonia sellowiana are positively photoblastic and reach the maximum percentage of germination at 23 ± 2°C in white light after seven days of imbibition. The pre-induction phase for spores induced by white or red light for 24 hours was 72 hours. Gametophytes grown in white light were plane and bidimensional, while those grown under red light were filamentous. The higher the number of hours of light applied per day during 10 days, the higher the percentage of germination. Germination was higher for long white light treatments applied on a daily basis. The effect of different light intensities on germination was also investigated here. The lower percentages of germination were observed for spores kept under 43% and 2% of full sunlight, while those kept under 26, 19 and 4% presented higher percentages. Spores presented circa 82% of germination after 731 days of storage under refrigeration at aproximately 10°C.

RESUMO - (Luz e armazenamento na germinação de esporos de Dicksonia sellowiana (Presl.) Hook., Dicksoniaceae). Os esporos de Dicksonia sellowiana são fotoblásticos positivos e atingem a máxima porcentagem de germinação a 23 ± 2°C em luz branca após sete dias de embebição. A fase de pré-indução para esporos induzidos por 24 horas de luz branca ou vermelha foi de 72 horas. Os gametófitos crescidos em luz branca apresentaram-se planos e bidimensionais enquanto os crescidos sob luz vermelha mostraram-se filamentosos. Quanto maior o número de horas diárias de luz aplicadas durante 10 dias, maior foi a porcentagem de germinação. A germinação foi maior em tratamentos diários de luz branca de longa duração. O efeito de diferentes intensidades luminosas sobre a germinação também foi estudado neste trabalho. Verificou-se que 43 e 2% da luz solar máxima produziram as menores porcentagens de germinação, enquanto que 26, 19 e 4% produziram porcentagens de germinação mais altas. Esporos apresentaram cerca de 82% de germinação após 731 dias de armazenamento sob refrigeração a aproximadamente 10°C.

Key words - Fern germination, gametophytes, light, storage

Introduction

Dicksonia sellowiana (Presl.) Hook. is an endangered tree fern which belongs to the Dicksoniaceae family. Its distribution ranges from southern Mexico through Central America and South America from Venezuela to Colombia, South to Bolivia, Paraguay, Uruguay and Southeastern Brazil. It grows generally in wet mountain in the tropics (Tryon & Tryon 1982, Sehnem 1978). In Brazil the tree trunks are cut and used to make jars and soil using in gardening to produce ornamental plants. A little is know about the germination and initial gametophytes growth of this species. Borelli et al. (1990) studied the fern propagation from spores of Cyathea schanschin Mart. and Dicksonia sellowiana (Presl.) Hook. These authors cultured spores in vivo and in vitro to produce gametophytes and sporophytes. There was a high percentage of germination and prothalli formation in Jones and Knop media, tree-fern fibre and Sphagnum moss substrates. Germination of several light sensitive spores is promoted by red light and the effect of red light is reversed by far-red light, implicating phytochrome control of the process (Raghavan 1993). Light is a requeriment for the germination of Platycerium bifurcatum (Cav.) C. Chr. (Camloh 1993), some species of Asplenium (Pangua et al. 1994) and Lygodium heterodoxum Kunze (Pérez-García et al. 1994) among a wide range of species belonging to different families. The photosensitivity of spores develops during imbibition under aerobic condition (pre-induction phase) and the photoinduction leads to cell division and protrusion of a rhizoid in the post-induction phase (Towill & Ikuma 1975). Spores of another tree fern, Cyathea delgadii Sternb., are positively photoblastic and the greater percentage of germination was obtained after a pre-induction of 48 hours of darkness and photoinduction of 1 minute red light (Randi & Felippe 1988a). This paper analised effects of white and red light treatments, light intensities and storage on germination of spores of Dicksonia sellowiana.

Material and methods

Sporophylls of Dicksonia sellowiana (Presl.) Hook. harvested on August 1995 and March 1996 in "Reserva Particular do Patrimônio Natural de Caraguatá", Antônio Carlos, Santa Catarina state, Brazil, were air dried in an oven at 30°C for 2-3 days. The spores were removed and separated from sporangia by filtering through lens paper after which they were stored in glass jars under refrigeration at approximately 10°C according to Randi & Felippe (1988a). Sterile culture conditions were used all over this work. Spores were treated with a 5% solution of commercial bleach (2% of active chlorine) for 10 minutes before being filtered through sterile filter paper and washed several times with sterile distilled water according to Randi & Crozier (1991). The spores were removed from the filter and placed in conical flasks containing liquid medium and 100 units nystatin.ml^_1 (Dyer 1979). The germination was carried out in growth cabinets at 23 ± 2°C. White light was provided by a 9W Energy Saving Desk Lamp which was compared to a 45W incandescent lamp and light intensity of 616 lux. The red light was obtained with a 15W fluorescent tube Sylvania Aquastar and a red cellophane paper filter, and light intensity of 194 lux. During dark treatments the conical flasks were covered with aluminium foil. The effect of different light intensities on germination was carried out at environmental conditions in 1m³ boxes covered with "sombrite" which cut full sunlight to 43%, 26%, 21%, 19%, 4% and 2% .The effect of storage at aproximately 10°C was analysed for the sample collected in July 1995. Spores where kept under refrigeration in glass jars during 15, 62, 119, 150, and 731 days. Duplicated treatments were carried out and two slides were prepared from each treatment with 100 spores counted on each slide. Spores were considered to have germinated after protrusion of the rhizoid. Before the experiments, the amount of normal spores were analised. Only normal spores were considered. Empty spores were not scored. Photomicrographies were carried out with fresh material in a Zeiss photomicroscope. The data were analysed by Excel for Windows (Microsoft) and Statgraphics softwares.The results are expressed in percentage but they were transformed to arc sin (%)^-0,5. To compare data, the LSD_5% (Tukey) and the confidence intervals at 95% were determined according to Snedecor (1962).

Results and Discussion

Dicksonia sellowiana presents nongreen spores. The germination was induced only by light and 48% of spores were empty. Spores of Lygodium heterodoxum Kunze show also positive photoblastism but the germination was promoted by white light and far red light (Pérez-García et al. 1994). Maximum germination of Dicksonia sellowiana in light occurred after seven days of imbibition at 23 ± 2°C for spores stored during 15 days under refrigeration (figure 1). Similar behavior was seen for Cyathea delgadii Sternb. (Randi & Felippe 1988b), Acrostichum danaeifolium Langsd. & Fisch. (Randi 1996), Asplenium ruta-muraria L., Asplenium trichomanes L. and Asplenium scolopendrium L. (Pangua et al. 1994).

Figure 1. Germination curve of D. sellowiana spores under continuous white light at 23 ± 2°C. Bars show confidence intervals at 95%. No germination ocurred in darkness.

In order to study the pre-induction phase and induction by white ligh and red light, spores of Dicksonia sellowiana were treated with 24 hours of white light or red light after 2, 4, 8, 12, 18, 24, 48, 72 and 96 hours of darkness. Some spores were able to germinate after only two hours of imbibition in darkness before 24 hours of the light treatments, but the percentage of germination reached the maximum after 72 hours of imbibition in darkness before 24 hours of light treatments (figure 2). Spores of Cyathea delgadii treated with one minute red light reached the maximum germination after a pre-induction of 48 hours (Randi & Felippe 1988a) while for spores of Acrostichum danaeifolium treated with 24 hours of white light the higher percentage of germination was seen after 48 hours of imbibition in darkness (Randi 1996). The red light treatment was more efficient than white light to promote germination of spores of Dicksonia sellowiana only after 24 hours of imbibition. Figures 3 to 6 show aspects of gametophytes of Dicksonia sellowiana grown under white and red light after seven and 15 days. Young gametophytes grown under white light are bidimensional and plane while the young gametophytes grown in the red light are filamentous. According to Raghavan (1993), filamentous growth is induced and sustained in the protonema grown in red light characterized by low photosynthesis, or in complete darkness, while its transition to the plane form is promoted by blue light which seems to be involved in the translation of some specific mRNAs.

Figure 2. Germination of D. sellowiana spores after different periods of imbibition in darkness and induction by 24 hours of white light (empty columns) and red light (black columns), 23 ± 2°C. Germination was scored after 10 days of imbibition. Different letters show statistically different results.

Figures 3-6. Gametophytes of D. sellowiana grown after seven days (3 and 4) and 15 days (5 and 6) of imbibition in constant white light (3 and 5) and in constant red light (4 and 6) at 23 ± 2°C.

The effect of daily white light treatments was also investigated in this paper (figure 7). The germination of spores of Dicksonia sellowiana was promoted by a single long white or red light treatment and by daily white light treatments. The greater percentage of germination was seen for spores treated with single long light treatment and long daily light treatment. Similarly, there was a positive correlation between germination and number of red light treatments given at 24 hours intervals with intervening periods of darkness for spores of Cyathea delgadii (Randi & Felippe 1988a). Spores of Acrostichum danaeifolium which received 12 hours of white light every day showed the same percentage of germination observed in continuous white light (Randi 1996). Furuya (1985) suggested that the transformation of Pr to Pfr would be sinchronic and gradual in a spore sample but some spores are able to germinate with low percentage of Pfr and some only with high percentage. According to Haupt & Psaras (1989) some transmitter Q is produced as long as Pfr is present in the spore and Q has to surpass a threshold in order to start germination in the spore. The distribution of responsiveness in the population, then could mean that each individual spore has a different threshold level or that the production of Q as a function of Pfr action time is the variable factor in the population.

Figure 7. Effect of daily white light treatments on the germination of D. sellowiana spores at 23 ± 2°C. Germination was scored after 10 days of imbibition. Different letters show statistically different results.

The effect of different light intensities (43%, 26%, 21%, 19%, 4% and 2% of full sunlight) in environmental conditions was also analised in this paper (table 1). It was observed that the spores of Dicksonia sellowiana were able to germinate in all the light intensities studied here, but the higher percentages were seen for the intermediary light intensities and the lower germination for the higher and the lower light intensities. In Dicksonia sellowiana the lower light intensity could reduced the percentage of germination by delaying the reversion of Pr to Pfr. The effect of red light fluence on conversion of Pr to Pfr was investigated in Lygodium japonicum L. (Tomizawa et al. 1983). The authors shown a clear positive correlation between % Pfr levels and germination. Higher light intensities contain proporcionally higher levels of blue and red light. Sugai et al. (1984) and Sugai & Furuya (1985) observed an inhibition of the red light promotion of fern spore germination by blue light. In Cyathea delgadii spores, a blue light treatment promoted less than 2% of germination while similar red light treatment promoted 50% of germination (Randi & Felippe 1988c). Spores of Dicksonia sellowiana which were kept in 43% of full sunlight received higher quanta flux of blue and red light and the blue light could cause some inhibitory effect over germination. On the other hand blue light seems to be necessary for the normal development of gametophytes. Notwithstanding, the blue light effect on germination was not analysed in this paper. Germination and gametophyte development seem to be dependent on the light quality and the light intensity, with red light promoting germination through phytochrome system and gametophyte growth being dependent on photosynthesis by chloroplast and RUBISCO synthesis.

The effect of different periods of storage under refrigeration in the germination of Dicksonia sellowiana is shown in table 2. Spores of Dicksonia sellowiana were still viable after 731 days of storage, although there was a small decrease in the percentage of germination after this period. These data suggest that this species could form spore bank in the soil. Spores of Cyathea delgadii kept under refrigeration at 3 ± 1°C did not show decrease in the percentage of germination after 275 days of storage under refrigeration (Randi & Felippe 1988b).The germination of Pteris vittata L. spores was lower after storage at room temperature with the storage period varying between 10 to 100 days (Beri & Bir 1993). Spores of Acrostichum danaeifolium stored during approximately two years at 3 ± 1°C presented circa 50% decrease in the percentage of germination (Randi 1996). On the other hand, green fern spores have the characteristics of recalcitrant seeds, as seen in Lygodium heterodoxum which when stored in dry conditions lose viability faster than when stored fully water-imbibed (Pérez-García et al 1994).

Acknowledgements- We thank Paulo Sérgio Schveitzer, manager of the Reserva Particular do Patrimônio Natural de Caraguatá for his kindness and facilities in supplying the material used throughout this work and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq- processo 523.623/94-1) for financial support.

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1. Departamento de Botânica, Universidade Federal de Santa Catarina, Caixa Postal 476, 88040-900 Florianópolis, SC, Brazil.

Departamento de Botânica, Universidade Federal de Santa Catarina, Caixa Postal 476, 88040-900 Florianópolis, SC, Brazil.

Publication Dates

• Publication in this collection
  19 Aug 1999
• Date of issue
  Apr 1999

History

• Received
  20 Apr 1998
• Accepted
  18 Sept 1998